Among glycoproteins, high molecular glycoprotein compounds, in which a sugar chain comprising approximately one to ten monosaccharides is bound via an O-glycoside bond at regular intervals to a peptide chain having a simple repeat structure, are collectively called mucins. Various mucins are present in cells or as components in plant and animal mucus in the natural world and are known to play various important roles in living systems. Moreover, mucins from plants and animals and contained in mucus components in foods are known to give important biological effects in life activity or in digestion and absorption processes, even when ingested as foods.
Approximately ten kinds of mucins have been identified in humans to date. These mucins are distributed and present mainly in mucosal portions such as saliva and gastric mucosa. Mucosal tissues formed by these mucins exhibit biological roles such as antibacterial effects as an extracellular matrix, by which viral infections or the like are blocked, in addition to physical effects such as the moisture retention, protection, and lubrication of cells and tissues (H. Nakata, Diversity of Mucin and Mucin-type Sugar Chain and Its Meaning: Understandable Glycobiology in Post-Genomic Era, Wakaru Jikken-Igaku Series (Understandable Experimental Medicine) (in Japanese), N. Taniguchi ed., Chapter 3, Yodosha Co., Ltd., 2002; K. Hotta, K. Ishihara, Search for Attractiveness of Gastric Mucus: Elucidation of Mucin using Newest Approach (in Japanese), Medical View Co., Ltd., 1999).
The physiological effects of these mucins do not always result from specific chemical reactions. Their physiological effects are also considered to be derived from their physical properties as substance, i.e., morphology including plasticity, viscosity, moisturizing properties, and so on, and from their ability to recognize a wide variety of molecules (e.g., lectin) due to the amorphous sugar chain portion bound to the peptide chain having a three-dimensional structure. Thus, the physical properties and three-dimensional structure of the polymer portion comprising of the peptide chain of mucins as well as the ability of molecular recognition by the amorphous sugar chain portion are needed to exert their functions.
On the other hand, such compounds constituting partial or main components in mucosa or an extracellular matrix exert their effects even when ingested from outside. Therefore, these compounds have been considered currently to have a great advantage that they may be artificially produced and supplied to the market as pharmaceuticals, cosmetics, foods, and so on (JP 8-269091A (1996)). Among sugar chain compounds, ahead of all others, chondroitin, chondroitin sulfate, and hyaluronic acid, etc., main components of an extracellular matrix, have been extracted and purified from various raw materials and provided into the market as foods, pharmaceuticals, cosmetics, and so on. However, mucins are taken merely dietary from, for example, some foods (aroid, okra, and Jew's-ear) or animals (cattle and pigs) (see JP 7-33623A (1995); JP 8-256788A (1996); JP 6-199900A (1994); JP 5-310799A (1993); and JP 7-126292A (1995)), and have not been supplied yet as compounds on a large-scale basis and in large amounts.
The glycoproteins including mucins have the molecular recognition ability and are expected to be useful in various use such as in pharmaceuticals. Nevertheless, an appropriate method for synthesizing them has not been found. In some cases, genes encoding the peptide sequences have been identified. However, approaches such as gene transfer or cloning have been attained with little success due to the difficulty for introduction of sugar chains after peptide chain synthesis (Polysaccharide Separation/Purification Method, Biological and Chemical Experimental Methods 20 (in Japanese), edited by K. Matsuda, Japanese Scientific Societies Press, 1987). For most glycoproteins, their synthetic methods have no excepting an approach involving synthesizing only the peptide chain by use of E. coli or the like and sequentially introducing sugar chains thereinto (see WO 96/13516). Such an approach has a disadvantage that they are unsuitable for large-scale production.
Glycoproteins include those with mucin-type sugar chains or those with asparagine-type sugar chains. Chaperone molecules which mediate binding of sugar chain are identified for some asparagine-type sugar chains, and binding sites of such sugar chains have been identified in some cases. Nevertheless, it is difficult to specify the sites of sugar chain introduction upon synthesis. Even if sugar chains can be introduced sequentially into an already synthesized peptide chain, it is expected that the higher order structure of the peptide chain is largely altered due to binding of sugar. Thus, there is no guarantee that the peptide chain forms the native higher order structure by refolding.
Meanwhile, restricted to mucin-type glycoproteins, the peptide chain forms a higher order structure by folding and then undergoes sugar chain modification. Therefore, the sugar chain can be bound to the peptide chain, with the maintained three-dimensional structure and functions of the protein. Thus, the sugar chain can be introduced with little loss of the whole higher order structure of the peptide chain (M. Fukuda, Mucin-type Sugar Chain, pp. 35-56, Y. Kohata, S. Hakomori, and K. Nagai ed., “Diverse World of Sugar Chain” (in Japanese), Kodansha Scientific, Ltd., 1993). Thus, mucin-type glycoproteins seems to have advantages in use for drug development. However, the amino acid sequences of binding sites in currently known mucin-type glycoproteins are not found to have any rule, and this makes it difficult to introduce a sugar chain at an intended position. Moreover, although the mucin-type glycoproteins have a relatively simple primary structure, it is also difficult to synthesize the whole mucin-type glycoproteins by a synthetic organic chemistry approach. For these reasons, it seems that an industrial approach for supplying mucin-type glycoproteins in large amounts has not been developed yet, although mucin-type glycoproteins have many superior characteristics.
A gel filtration method, also called Size Exclusion Chromatography (SEC), has been used widely for a long time as a convenient and accurate approach for measuring the molecular weights of polymer compounds. This method has been used not only as analysis using open columns but also as high-performance liquid chromatography and also allows fractionation based on molecular weights, particularly, automatic fractionation (A. Fallon, R. F. G. Booth, L. D. Bell, translated by T. Osawa, High-Performance Liquid Chromatography, Biochemical Experimental Method 9 (in Japanese), Chapter 5, Tokyo Kagaku Dojin Co., Ltd. 1989). However, it is technically difficult to determine the absolute value of the molecular weight of an unknown substance only by performing these measurements. Specifically, there are two requirements that a column carrier, with which it is assured that gel filtration can be done with good reproducibility according to a theoretical calibration curve, is used and that an accurate standard molecular weight marker is used. Thus, the combination of a test substance and a column carrier and the combination of a test substance and a molecular weight marker must be chosen sufficiently carefully.
A measurement method using a time-of-flight mass spectrometer (MALDI-TOF MS) has been spread in recent years as such an approach for absolute molecular weight measurement. This approach can achieve absolute measurement by which the molecular weights of polymer compounds are determined accurately. However, the apparatus for this method is much more expensive than liquid chromatographs. It is actually impossible to spread the apparatus into all chemical synthesis laboratories, factories, medical facilities, and so on. Analysis may be conducted centrally at one location in which the expensive equipment is placed or may be outsourced. However, laboratories, which require quick feedback and desire rapid measurement, still utilize analysis using SEC with frequency. In such a case, it is preferred that a common substance that can be measured by both MALDI-TOF MS and SEC should be used as a standard for absolute molecular weight measurement.
As long as the SEC approach is used, a standard substance used in the combination of a test substance and a molecular weight marker must be as similar in physical property to a test substance as possible. The principle of SEC is that separation is achieved on the basis of a solute size (molecular weight) by use of molecular sieve effects brought by a polymer filler network. Therefore, the separation depends on physical properties such as size or shape but not on chemical properties that give the interaction between the solute and a stationary phase. Specifically, substances similar in hydrodynamic radius and shape of a polymer in a solvent (mobile phase) need to be selected for use as the standard. SEC users commonly select and utilize, from catalogues, polymer molecular weight markers that take conformation as similar to one another as possible. However, for previously forming a marker that has narrow molecular weight distribution and has a molecular weight controlled to some extent, it is most convenient to use a synthetic polymer for which a method for controlling a polymerization process is known. Thus, a very limited number of substances are commercially available as molecular weight markers. Specifically, only polymers having a linear structure, such as polystyrene, polymethyl methacrylate (PMMA), polyethylene, polyethylene glycol, polyethylene oxide, polyacrylic acid, and pullulan, are now on the market (e.g., JP Patent No. 3012917 (JP 10-60005A (1998))). Under such circumstances, it is impossible to cover all of many polymer compounds.
Among others, glycoproteins (e.g., enzymes, mucins, and hormones) whose physiological actions have received attention in recent years have no appropriate standard molecular weight markers. The glycoproteins are universally distributed in the natural world and are present in larger numbers than proteins free of sugars. Some of them have plural sugar chains bound to the peptide chain and exhibit a brush-like form, while glycoproteins with only one sugar chain bound per molecule are present and even these have a very bulky sugar chain portion covering the surface of the molecule. When such a glycoprotein is analyzed using SEC for separation on the basis of a “molecular size and shape”, it is obvious that the use of conventional molecular weight markers having a linear structure is inappropriate. For example, pullulan, a polysaccharide, has been used as a molecular weight marker for such a reason that it contains sugars. However, there has been no guarantee so far that such a molecular weight marker provides an accurate molecular weight. Under present circumstances, the evaluated molecular weight, which may however be wrong, only indicates the relative relationship with other markers used. Specifically, the estimation of molecular weights only by SEC was basically inaccurate and required confirmation using another method.
Electrophoretic methods such as SDS-PAGE are also protein separation analysis approaches that can be used conveniently in laboratories. Appropriate molecular weight markers may also be needed and are generally used for such analytical approach, as in SEC. Such molecular weight markers are also used in the fields of various common biochemical analyses other than SEC and electrophoretic methods.
Jellyfishes, that are seen predominantly in the summer period, sometimes are seen in a large number and may therefore significantly lower the efficiency or economic effects of the intake/drainage system in nuclear power or thermal power plants, of the intake/drainage system for industrial water in a variety of factories facing the ocean, of harbors, of fishery with fishing nets such as fixed shore nets, and so on. Particularly, moon jelly (Aurelia aurita) or the like, which has a poor swimming ability, must be eradicated actively, particularly when seen in a large number. Large jellyfishes such as Echizen-kurage jellyfish (Nemopilema nomurai), when seen in a large number, require, due to their weights, massive operation for pulling them up for evacuation from the ocean using heavy machineries or the like. As a result of such operations, jellyfishes are pulled up from the ocean in large amounts at once. However, the jellyfishes once pulled up are regarded as wastes under current Japanese law and prohibited from being disposed of again into the ocean. Therefore, they must be landed and accumulated. Methods for utilizing such accumulated jellyfishes as foods or as fertilizers have been proposed (e.g., JP 2004-99513A; JP 2003-321497A; JP 2001-178492A; JP 2002-370991A; WO 95/17428; JP 2002-143824A; JP 6-217737A (1994); and V. Schmidt, A. Bally, K. Beck, M. Haller, W. K. Schlage, C. Weber “The extracellular matrix (mesoglea) of hydrozoan jellyfish and its ability to support cell adhesion and spreading. Hydrobiologia 216-217, pp. 3-10 (1997)). However, due to the absence of other effective ways to use them, disposal of them for the reason of environmental protection places an enormous economic burden on corporations or municipalities in charge. For obtaining costs for disposal, it is desired that costs for promoting the disposal of the residue should be recovered by isolating expensive valuables from them even in small amounts. However, effective solutions therefor have not been obtained yet.
The amount of moon jelly (Aurelia aurita) seen in a large number is estimated by air observation or the like and allegedly reaches several hundreds of thousands of tons per gulf in some cases (T. Yasuda ed., “Marine UFO Jellyfish” (in Japanese), pp. 41-77 VII Emergence and Distribution, Kouseisha Kouseikaku Co., Ltd., 2003). Since jellyfishes are present as marine resources with rich abundance on Earth, not only are the accumulated wastes used, but the utilization thereof by active harvest can be taken into consideration.